Activities of LL-37, a Cathelin-Associated Antimicrobial Peptide of Human Neutrophils

Abstract

Human neutrophils contain two structurally distinct types of antimicrobial peptides, ␤-sheet defensins (HNP-1 to HNP-4) and the ␣-helical peptide LL-37. We used radial diffusion assays and an improved National Committee for Clinical Laboratory Standards-type broth microdilution assay to compare the antimicrobial properties of LL-37, HNP-1, and protegrin (PG-1). Although generally less potent than PG-1, LL-37 showed considerable activity (MIC, <10 g/ml) against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, and vancomycin-resistant enterococci, even in media that contained 100 mM NaCl. Certain organisms (methicillin-resistant S. aureus, Proteus mirabilis, and Candida albicans) were resistant to LL-37 in media that contained 100 mM NaCl but were susceptible in low-salt media. Burkholderia cepacia was resistant to LL-37, PG-1, and HNP-1 in low-or high-salt media. LL-37 caused outer and inner membrane permeabilization of E. coli ML-35p. Chromogenic Limulus assays revealed that LL-37 bound to E. coli O111:B4 lipopolysaccharide (LPS) with a high affinity and that this binding showed positive cooperativity (Hill coefficient ‫؍‬ 2.02). Circular dichroism spectrometry disclosed that LL-37 underwent conformational change in the presence of lipid A, transitioning from a random coil to an ␣-helical structure. The broad-spectrum antimicrobial properties of LL-37, its presence in neutrophils, and its inducibility in keratinocytes all suggest that this peptide and its precursor (hCAP-18) may protect skin and other tissues from bacterial intrusions and LPS-induced toxicity. The potent activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it or related molecules might have utility as topical bronchopulmonary microbicides in cystic fibrosis.

Structural features and biological activities of the cathelicidin-derived antimicrobial peptides

“…22,26,36,46,78,80,81 In aqueous buffer peptides are in general unordered, but they show a transition to an ␣-helical conformation in the presence of the helicogenic solvent trifluoroethanol, 22,26,36,78,80,81 or in anisotropic environments such as SDS micelles 82 and phospholipid liposomes, 82 or in the presence of lipid A. 82,83 Exceptions are PMAP-37 and LL-37/hCAP18, which assume a helical conformation also in aqueous solutions, respectively, at neutral pH 28 or in the presence of anions such as bicarbonate and sulfate. 84 This behavior has been attributed to concentration-dependent oligomerization, which may be induced by ionic and hydrophobic interactions favored respectively by the presence of several negatively charged residues in both peptides and of a hydrophobic N-terminal region in LL-37/hCAP18.…”

“…22,26,28,82 Structural analysis by two-dimensional (2D) nmr spectroscopy has shown that the rabbit peptide CAP18 106 -137 is in a rigid, completely ␣-helical conformation in the presence of 30% trifluoroethanol, 83 and Fourier transform infrared (FTIR) spectroscopy indicates that LL-37/hCAP18 has a helical content of 80 -85%, in the presence of acidic or zwitterionic membranes. 85…”

“…22,27,28,46,78,81,82,85 Some of them, particularly those with C-terminal (BMAP-27, BMAP-28, and SMAP-29) or N-terminal (LL-37/ hCAP18) hydrophobic regions, are also toxic to eukaryotic cells, 22,81,84,85 while others, such as PMAP-23, CRAMP, and BMAP-34, are much less or not at all effective. 27,46,78 Targets include normal and tumor cell lines and red blood cells, 22,84 and the effect is often exerted at concentrations that are only slightly higher than those active against microorganisms.…”

“…89 Similar results have been observed with human LL-37/ hCAP18 and its fragments. 37,82 Synthetic fragments of BMAP-27, BMAP-28, and LL-37/hCAP18, lacking their hydrophobic region, show an improved selectivity, with a significant reduction in cytotoxic activity to eukaryotic cells and an unchanged or only modestly decreased antibacterial activity. 22,84,85 The role of the hydrophobic region has further been investigated in BMAP-28.…”

“…The antimicrobial activity of protegrins, particularly PG-1, was studied against a wide panel of microorganisms (examples are given in Table II). These peptides display a potent and broadrange activity against a number of gram-negative bacteria 82,157 including Neisseria gonorrhoeae 158 and Chlamydia trachomatis, 159 gram-positive bacteria, such as MRSA and VREF strains, 82,157 Mycobacterium tuberculosis, 160 fungi, 161 and enveloped viruses. 162 Protegrins also proved active against several gram-negative peridontal pathogens, with a much higher potency than that of other antimicrobial peptides.…”